Technetium bone scanning medium

ABSTRACT

AN AQUEOUS SOLUTION OF ETHANE-1-HYDROXY-1,1-DIPHOSPHONATE (EHDP) IS ADDED TO AN ACIDIC SOLUTION OF STANNOUS CHLORIDE ND MIXED. 99MTCO4 IN ISOTONIC SALINE SOLUTION IS THEN ADDED AND HE MIXTURE IS AGAIN MIXED. THE SOLUTION IS ADJUSTED TO A PH OF ABOUT 6-7 YIELDING A PREPARATION APPROPRIATE FOR INJECTION INTO A SUBJECT. THE MEDIUM IS HIGHLY SPECIFIC WITH RESPCT TO DEPOSITION IN THE SKELETON WHILE TROUBLESOME AND UNDESIRED EFFECTS SUCH AS COLLOID FOMATION, BINDING TO BLOOD PROTEINS, LOCALIZATION IN THE KIDNEYS, ETTC., ARE AVOIDED.

United States atent O 3,735,001 TECHNETIUM BONE SCANNING MEDIUM JamesMcRae, El Cerrito, Donald C. Van Dyke, Berkeley, and Yukio Yano,Richmond, Calif., assignors to the United States of America asrepresented by the United States Atomic Energy Commission No Drawing.Filed May 24, 1972, Ser. No. 256,233 Int. Cl. A61k 27/04 US. Cl. 424-1 9Claims ABSTRACT OF THE DISCLOSURE An aqueous solution ofethane-l-hydroxy-l,l-diphosphonate (EHDP) is added to an acidic solutionof stannous chloride and mixed. TcO in isotonic saline solution is thenadded and the mixture is again mixed. The solution is adjusted to a pHof about 6-7 yielding a preparation appropriate for injection into asubject. The medium is highly specific with respect to deposition in theskeleton while troublesome and undesired effects such as colloidformation, binding to blood proteins, localization in the kidneys, etc.,are avoided.

BACKGROUND OF THE INVENTION The invention herein was made under, or inthe course of Contract No. W-7405-ENG-48 with the United States AtomicEnergy Commission.

Radio-scanning and similar radiographic techniques are findingever-increasing application in biological and medical research anddiagnostic procedures. Radio-scanning procedures involve the preparationof radioactive media which, upon injection, infusion or are otherwiseintroduced into a biological subject, become localized in specificorgans, tissue or skeletal material which are under study. When solocalized traces, plots or so-called photographs of the distribution ofthe radioactive material may be made by various radio-scanning means,i.e., such as by radiation detector arrays, traversing scanners,scintillation cameras and the like. See, for example, Nuclear Physics inMedicine, Gordon L. Brownell and Robert J. Shalck. Physics Today, August1970, pp. 32-38. The resultant distribution and corresponding relativeintensities may then be used to indicate the position occupied by thetissue in which the medium is localized as well as to indicate apresence of aberrations, pathological conditions and the like. Thefollowing references disclose radioscanning devices as used in the art.

Anger, H. 0.: While-body scanner Mark II. J. Nucl. Med.

Anger: H. 0.: Tomographic gamma-ray scanner with simultaneous readout ofseveral planes. In Fundamental Problems in Scanning, Gottschalk, A. andBeck, R. N., eds., Charles C. Thomas, Sprinfield, 111., 1968.

The applicability of bone scanning techniques is limited largely by theavailability of radio-scanning media of sufficient specificity andhaving an appropriate radiation emmission. Radio-scanning agents usedfor bone scanning heretofore leave much to be desired. A need thereforexists for improved bone (skeletal tissue) radio-scanning media.Technetium-99m possesses radio-emissive properties which would besuitable for such use provided that an injectable preparation havingspecific bone-localization properties were available.

SUMMARY OF THE INVENTION The present invention relates, in general, toradioscanning procedures and, more particularly, to preparation and useof a radio-scanning composition includingethane-l-hydroxy-l,l-diphosphonate combined with technetium Tc) throughmediation by a stannous chloride reagent as well as to the compositionitself.

Accordingly, it is an object of the invention to provide a superioragent for use in radiographic skeletal bone-scanning procedures.

Another object of the invention is to provide a composition includingtechnetium labelled ethane-l-hydroxy- 1,1-diphosphonate for use as aradiographic bone-scanning medium.

Still another object of the invention is to provide for the preparationof a composition including ethane-l-hydroxy-l,1-diphsophonate carryingtechnetium in a form especially adapted to preferentially localize inskeletal bone tissue for injection into biological subjects and forsubsequent scanning to display said bone tissue.

Other objects and features of the invention will be apparent in thefollowing description:

For preparing the radio-scanning medium of the invention there is firstprepared a fresh dilute solution of stannous chloride in dilutehydrochloric acid. For convenience relative proportions comprising about10 mg. of reagent grade SnCI -ZH O which may be dissolved in about 100ml. of 0.01 N HCl with thorough mixing. A portion of such solutionrequired for one or more doses may then be filtered through anultra-filter medium (e.g., an 0.22 1. Millipore filter) to removebacteria and other extraneous materials, to be deposited for example,into an evacuated vial or a container containing inert gas so as toexclude oxygen.

A corresponding proportion of ethane-l-hydroxy-l,1- diphosphonate(EHDP), i.e., about 5 mg. of EHDP in about 1 ml. of sterile oxygen freewater is dissolved with thorough mixing. The EHDP solution is then addedto the stannous chloride solution with mixing.

Then the desired quantity of TcO activity is dissolved in normal salinesolution with mixing. A relative proportion of about 3 ml. of salinesolution may be used. Commercially available MEK (methyl ethyl ketone)extracted TcO having a concentration of 20 mCi/ml. in isotonic salinesolution, or other suitable preparation, may be used to furnish theradio-technetium. For the relative volumes given herein, abotu 2.5 ml.to about 3.75 ml. of the foregoing commercial technetium activity may beused. An aliquot portion of the foregoing solution containing theappropriate dosage of TcO, is then added to and mixed with the EHDPsolution containing SnCl -2H O. Thereafter, the pH of the solution isadjusted to a value in the range of about 6 to 7 with dilute alkali suchas NaOH solution. The EHDP- Tc solution preparation may then be filteredthrough an ultra-filter such as an 0.22 a Millipore filter to insuresterility and remove possible pyrogenic substances.

The resulting EHDP- Tc preparation is carrier free and containsessentially about 1 mg. EHDP/ml, 0.02 mg. SnCl -2H O/ml. and about 10-15mCi Tc/ml. The procedure insures that at least about 95% of thetechnetium activity is chemically bound to the EHDP, i.e., the chemicallabelling efficiency is typically about 95% of the TcO added to thepreparation. The binding efficiency and stability of the preparationwere determined by ascending paper chromatography using Whatman No. 1filter paper strips in methanol solvent.

To insure longer shelf life the reagent solutions should be made oxygenfree as by saturating the solutions with substantially oxygen freenitrogen gas and performing the operations in an oxygen free atmosphereor in a vacuum, e.g., a sealed evacuated serum vial. The reagents andglassware should be sterile and pyrogen free.

Preparations made by the foregoing procedure, may then be injected intosuitable subjects, with the result that adequate amounts of thetechnetium activity are directed to localize principally in skeletaltissue (bone) to then permit radio-scanning using equipment andprocedures known in the art. (Table I).

It is to be particulraly noted that variations in the method ofpreparation of the compound such as increased concentrations of Sn(I-I)and EHDP and altering the order of addition of the reagents results inpreparations that localize primarily in the kidneys with retention ofthe activity in the kidneys even after 24 hours. The results ofincreased concentrations of EHDP (1O m./ml.), SnII (0.2 rug/ml.) andSn(II), TcO EHDP order of addition of reagents is shown in Table 2. Onthe other hand, when the described method of preparation was followed,approximately 6070% of the radioactivity was excreted by way of thekidneys. This excretion was nearly complete at 3 hours. There was littleretention of activity within the kidneys which were thusly only faintlyvisualized by scintillation camera pictures.

TABLE I Percent, uptake of EHDP Tc-Sn per Whole organ of rat for varyingmolar raltios of EHDP2S1'lCl2'12HzOThI6 hours after intravenousinjection Molar ratio EHDPzSnClrZIHO 1 :1, ml. 1 5021, ml. 3 10021, ml

Muscle femur 0.008

Marrow plus femur. 1. 08 1. 88 1.10

Marrow (by difi 0.07 0. Femur 1. 01 1. Careass 35. 7 39 1 Mean of 2rats, 0.5 mg. EHDP/kg. rat. 2 Mean of 4 rats, 5 mg. EHDP/kg. rat. 3 Meanof 2 rats, 5 mg. EHDP/kg. rat.

TABLE II Percent uptake of EHDP- 'Ie-Sn per whole organ of rat for EHDP-Sn(II)- m'le and for Sn(II- Tc-EHDPPreparations in order of reagentaddition] EHDP- Sn(ID- EHDP- Sn(II) m1. ml. m1. ml.

t 0. 098 1. 72 0. 672 1. 41 Marrow plus femur- 2. 11 1. 22 1. 63 1. 13Marrow (by diff.) 0.13 0.06 Femur 1. 49 1. 07 C 30. 4 26. 8

1 livlean of 2 rats, 5.0 mg. EHDP/kg. rat, 3 hours post-intravenousinject on 1 Mean of 2 rats, 5.0 mg. EHDP/kg. rat, 20 hours post-TV.injection. 3 One rat, 5.0 mg. EHDP/kg. rat, 20 hours post-LV. injection.

Table III, infra, shows the results of diluting the standard EHDP- Tcpreparation 100-fold. The data indicates a breakdown of EHDP Tc in thediluted 4 preparation with a decrease in bone uptake and an increase insoft tissue uptake.

TABLE III [Percent uptake of EHDP- Te per whole organ of rat forstandard 50:1, EHDPzSnII, for undiluted and times dilution ofpreparation] Undiluted 1 dilution 2 Marrow plus femur 1. 65 0. 651Carcass 46. 4

1 5 mg. EHDP/kg. rat. 2 .05 mg. EHDP/kg. rat. Nora-Time after injection,3 hours.

The blood disappearance curves can be approximated with at least threecomponents with T min., T 17 min., and T 12 min.

The method of using Sn(II) for labelling EHDP with Tc is dependent uponthe ability of chelating agents to stabilize Sn(II) and increase itsusefulness as a reducing agent for TcO. Furthermore, the stability ofS11 TC-EHDP preparation in vivo minimizes colloid formation at neutralpH and also minimizes the binding of Sn- Tc to blood proteins.

The stability of the EHDP- Tc in vitro is dependent upon removing air,i.e., oxygen, from the preparation. The initial chemical bindingefficiency ranges from 90- 96% and the amount of Tc still bound to EHDPafter 6 hours is 85-90% in an O -free system. When 0 is not excluded,the preparation breaks down within 2-4 hours to about 6575% Tc bound toEHDP.

The estimated radiation dose delivered to a 70 kg. patient from 10 mCiof EHDP- Tc is:

D (whole body) =0.148 rad. D (skeleton)=3.68 rad.

D (kidneys)=3.68 rad.

D (bladder)=7.36 rad. D=Dosage.

Materials and Methods Commercially available MEK extracted T00 (20mCi/ml. in isotonic saline is used for the preparation of Sn- Tc-EHDP,hereafter referred to as EHDP- 99mT The method for making EHDP- Tc is asfollows:

(1) Dissolve 10 mg. of reagent grade SnCl -2H O in 100 ml. of 0.01 N HClacid and filter 1 ml. of this solution through a 0.22 Millipore filterinto an evacuated serum vial. Reagent grade SnCl -2H O is used andfreshly prepared in 0.01 N HCl acid just prior to the labellingprocedure.

(2) Dissolve 5 mg. of EHDP in 1 ml. of sterile 0 free H 0 and add to thel-ml. Sn(II) and mix for 1-2 minutes. The EHDP used as in this exampleis available through the Procter and Gamble Co. under the trade nameEHDP.

(3) Add the desired activity of TcO in 3 ml. of saline and mix for 1-2minutes. I

(4) Adjust the preparation of pH 6-7 with dilute NaOH solution. 1 p

(5) Finally filter the EHDP- Tc through a 0.22 Millipore filter toinsure sterility of the preparation.

The 'EHDP- Tc preparation is To fcarrier free and contains 1 mg.EHDP/ml. 0.02 mg. SnCl -2H O/ml. and 10-15 mCi Tc/ml. The chemicallabelling efficiency is about 95% of the TcO added to the preparation.

The binding efliciency of the chemical labelling procedure and thestability of the preparation were determined by ascending paperchromatography 'using Whatman #1 paper strips in 85% methanol solvent.

To insure longer shelf life, of EHDP- Tc, the reagent solutions shouldbe made free of dissolved oxygen by saturating the solution withnitrogen and performing the chemical preparation in an evacuated andsealed serum vial. The reagents and glassware used for the preparationare sterile and pyrogen free.

Evaluation of EHDP- Tc in animals The EHD P- Tc compound was evaluatedby injection into Sprague-Dawley rats weighing 200-250 grams and inbeagle dogs Weighing about 20 pounds. In rats 0.5 ml. of the materialwas injected intravenously by tail vein, scintillation camera pictureswere obtained to visualize the distribution of EHDP Tc at diiferenttimes after intravenous injection. In rats both the multichannel and A5"pinhole collimators were used. In dogs the whole body scanner and/or thetomographic scanner were also used.

Tissue distribution studies were done in rats by administering 0.5-1.0ml. of EHDP- Tc and sacrificing the animals at 3 hours and 20 hours.

Blood disappearance half-times were determined in dogs by taking 1-ml.blood samples from 1 minute to 180 minutes after administration of theEHDP- Tc. The blood samples were counted in a Well counter using an NaI(Tl) crystal and the data were plotted on semilog paper to obtain theblood disappearance half-times. A known dilution of the injected dose ofTc was counted to determine the blood activity at zero time afterinjection.

Results and discussion Table I, supra, shows the percent uptake of EHDP-Tc for whole organs of rat (except skeleton which is represented byfemur uptake and blood which is shown as uptake per ml.) for :1, 50:1,and 100:1 molar ratios of EHDP to SnCl -2H O three hours afterintravenous injection. The uptake of Tc in the carcass is primarilyskeletal uptake and appears to be optimal at the 50:1 molar ratio ofEHDP:SnCl '2H O. Molar ratios over the range of about 30:1 to about 70:1may also be used. The percent uptake in the narrow was determined bydifference from femur-l-marrow counts/min. less the counts/ min. offemur alone.

While there has been described in the foregoing what may be consideredto be preferred embodiments of the invention, modifications within theskill of the art may be made therein and it is intended to cover allsuch as fall within the scope of the appended claims.

What We claim is:

1. In a process for preparing a Tc-ethane-i1-h'ydroxy-1,1-diphosphonateradio-scanning medium, the steps comprising:

mixing a dilute acidic aqueous solution of stannous chloride with adilute aqueous solution of ethane-l-hydroxy-1,1-diphosphonate;

adding a dilute saline solution containing Tc activity to the solutionderived in the foregoing step and into mixing same; and

adjusting the pH of the intermixed solution to a pH in the range ofabout 6 to 7 to yield said radio-scanning medium.

2. A process as defined in claim 1 wherein said stannous chloridesolution comprises a dilute solution of SnCl acidified with HCl andwherein the molar ratio of said ethane-l-hydroxy-l,l-diphosphonate tosaid SnCI -ZH O is in the range of about 30:1 to about :1.

3. A process as defined in claim 1 wherein said SnCl 2H O solutioncomprises about 0.01 N HCl and is prepared and stored in an oxygen freeenvironment.

4. A process as defined in claim 2 wherein the relative proportions ofthe components are such as to yield about 1 rng./ ml.ethane-l-hydroxy-1,1-diphosphonate, 0.02 mg./ ml. SnCl -2H O and about10 to 15 mCi/ml. of Tc/ml. in the final preparation.

5. A process as defined in claim 4 wherein said operations for preparingthe preparation and storage of the preparation are conducted in asubstantially oxygen free environment and the preparation is sterilizedby ultrafiltration.

6. A process as defined in claim 4 wherein the additional steps ofintroducing said final preparation into a subject and radio-scanning thesubject to determine the skeletal distribution of the Tc activity areincluded.

7. A radio-scanning injectable aqueous solution composition comprisingSnCl -2H O and ethane-l-hydroxy- 1,1-diphosphonate in molar ratioproportions in the range of about 30:1 to about 70:1 and with anappropriate quantity of Tc activity in association therein.

8. A radio-scanning composition as defined in claim 7 wherein said molarratio proportion is about 50:1.

9. A radio-scanning composition as defined in claim 7 comprisingessentially about 1 mg./ml. of ethane-l-hydroxy-1,1-diphosphonate, about0.02 mg./ml. SnCl -2H O and about 10 to about 15 mCi Tc activity, saidsolution having a pH value in the range of about 6 to 7.

References Cited UNITED STATES PATENTS 3,436,354 4/1969 Gemmill252--301.1 R 3,466,361 9/ 1969 Richards 424-1 3,468,808 9/1969 Arino252-301.1 R 3,683,066 8/1972 Ascanio et al. 424-1 BENJAMIN R. PADGETT,Primary Examiner U.S. C1. X.R.

250-7l.5 S; 252--30l.1 R

